A Firefly Luciferin
The visualization of a biological event and the phenomena have been considered to be important, and it is desired that the object of visualization is expanded. Thus the diversification of a labeling technique is desired. In particular, the labeling technique for molecular imagings has been developed along with a progress of an apparatus for a diagnosis and examination. For example, the labeling techniques for applying to the advanced technology such as personalized medicines for cancer or cardiac disease are examined intensively. The needs for apparatuses and labeling materials with higher-sensitivity and higher-performance are also rapidly increasing, according to the measurement technology.
As is well known, the bioluminescent system of firefly shows great efficiency in luminescent, and it is said that this system is one of the most efficiently systems that can convert energy into a light. The understanding of the molecular mechanism of the bioluminescence is in advance.
It is known that this bioluminescence of the firefly emit a luminescence by chemically reacting with the luciferin which is luminescent substrate through the carton of the luciferase which is luminescent enzyme. In this reaction, the luminescent substrate is adenylated (luciferyl-AMP) in the presence of an adenosine triphosphate (ATP) and divalent magnesium ion (Mg2+) lead to an adenylylated form which is substrate of an activated form. Then, a peroxide anion is produced by a luciferase of this form and converted into dioxetanone which is a high energy peroxide. The unstable dioxetanone dissolves itself and releases proton and carbon dioxide, and produce an excited singlet state of a monoanion form. Furthermore, this is protonated to produce an excited singlet state of a dianion form. It is assumed that the luminescence from the excited singlet state of a monoanion form is red. On the other hand, the luminescence from the excited singlet state of a dianion form is yellowish green, and this is assumed to be the luminescence of the firefly. The product after the luminescence is named oxyluciferin.
The luminescence of luciferin-luciferase reaction is very efficient and an elucidation of the molecular mechanism of the bioluminescence has been explained as described above. In light of these circumstances, the luminescent materials using the firefly bioluminescent system are commercially available from many companies. However, the research and development by the side of proteins (enzymes) have been done to a great extent, there are very few approaches from the side of a low molecular compound regarding to the luminescent materials relating to the firefly bioluminescence, because these materials have already been applied into practical use in the field of a medical and a biochemical field mainly. In particular, there are very few studies for correlation between a structure and an activity such as the structure of the luminescent substrate being converted.
Furthermore, the materials such as kit products, for labeling with luminescent using the firefly bioluminescent system are not inexpensive, although the luminescent enzyme can be provided at low cost by a recombination technique. This originates in that the luminescent substrate is the luciferin. The luciferin of D-form which is the luminescent substrate in nature is now synthesized by the D-cysteine which is not an amino acid of nature, which is one of the costly materials.
Needs and the Circumstance of the Luminescence of Multiple Colors Using the Bioluminescent System
The luminescence of multiple colors is desired in a detection system using a label in order to observe many events. Therefore, it is desirable that a width of a wavelength of the labeling materials which is available for the detection system is broader. The labeling materials which is luminescent in red is desired for the use in a deep labeling in vivo in that the long wavelength light has greater optical transparency than the short wavelength light. For example, it is desirable that the label have the luminescent ranging from less than about 450 nm-more than about 650 nm in preparing the labeling materials.
There are substrates having some luminescent wavelengths which are available as the luminescent substrate for the firefly bioluminescent system. The both ends of the shortest and the longest wavelengths of these substrates are blue of a coelenterazine system (about 480 nm) and red of the firefly system (about 613 nm). They are available and manufactured from the Promega Inc. Also, the luminescent material of longer wavelength in red using the luminescent enzyme of the Phrixothrix hirtus is commercially available recently from TOYOBO Co., Ltd. The prospective potential needs are expected not only for these luminescent wavelengths, but also for the further expansion of the both ends of the shortest and the longest wavelengths in the luminescent wavelengths.
The example of the existing products having red and blue luminescence utilizing the bioluminescent system are shown in the followings:
1. Promega Inc.: Chroma-Luc: 613 nm (Non-patent literature 1)
This system utilizes a mutant of the Click Beetles and wild-type firefly luminescent substrate.
2. TOYOBO Co., Ltd.: MultiReporter Assay System-Tripluc: 630 nm (Non-patent literature 2)
This system utilizes the red luminescent enzyme of the railroad worm and wild-type firefly luminescent substrate. The luciferase gene of a green luminescent luciferase (SLG, maximum emission wavelength 550 nm), a orange luminescent luciferase (SLO, 580 nm) and a red luminescent luciferase (SLR, 630 nm) in the color was used to change the color of luminescent. Is it the utilization of the luminescent enzymes that bring different color of the luminescence.
3. University of Tokyo: Aminoluciferin: About 610 nm (patent document 1)
This discloses a luciferin derivatives.
4. Promega Inc.: Chroma-Luc: about 480 nm (Non-patent literature 3)
This system utilizes a coelenterazine and Renilla reniformis luciferase.
5. ATTO Inc.: Vargula hilgendorfii bioluminescence, about 460 nm (Non-patent literature 4)
This system utilizes a substrate of the coelenterazine system and Renilla reniformis luciferase.
We also disclose a compound analogous to luciferin in patent document 2. These compounds are compounds which have the structure analogous to luciferin.    patent document 1: Japanese Patent Application KOKAI Publication No. 2007-091695    patent document 2: International publication No. 2007/116,687    Non-patent literature 1: Promega Inc. general catalogue 2007, 12.16    Non-patent literature 2: Upload vol. 79, 2005 p 1-10, Toyobo Biochemicals for Lifescience 2006/2007 p 4-67    Non-patent literature 3: Promega Inc. general catalogue 2007, 12.14    Non-patent literature 4: ATTO Inc. general catalogue 2008-2009p 247